In situ self-assembled organoid for osteochondral tissue regeneration with dual functional units.

The regeneration of hierarchical osteochondral units is challenging due to difficulties in inducing spatial, directional and controllable differentiation of mesenchymal stem cells (MSCs) into cartilage and bone compartments. Emerging organoid technology offers new opportunities for osteochondral regeneration. In this study, we developed gelatin-based microcryogels customized using hyaluronic acid (HA) and hydroxyapatite (HYP), respectively for inducing cartilage and bone regeneration (denoted as CH-Microcryogels and OS-Microcryogels) through in vivo self-assembly into osteochondral organoids. The customized microcryogels showed good cytocompatibility and induced chondrogenic and osteogenic differentiation of MSCs, while also demonstrating the ability to self-assemble into osteochondral organoids with no delamination in the biphasic cartilage-bone structure. Analysis by mRNA-seq showed that CH-Microcryogels promoted chondrogenic differentiation and inhibited inflammation, while OS-Microcryogels facilitated osteogenic differentiation and suppressed the immune response, by regulating specific signaling pathways. Finally, the in vivo engraftment of pre-differentiated customized microcryogels into canine osteochondral defects resulted in the spontaneous assembly of an osteochondral unit, inducing simultaneous regeneration of both articular cartilage and subchondral bone. In conclusion, this novel approach for generating self-assembling osteochondral organoids utilizing tailor-made microcryogels presents a highly promising avenue for advancing the field of tissue engineering.

Bibliometric and visualization analysis of stem cell therapy for meniscal regeneration from 2012 to 2022.

Background: Meniscus injuries, a common joint disease caused by long-term wear, trauma and inflammation, usually cause chronic dysfunction and pain in the joint. Current clinical surgeries mainly aim to remove the diseased tissue to alleviate patient suffering instead of helping with meniscus regeneration. As an emerging treatment, stem cell therapy has been verified to facilitate meniscus regeneration effectively. The purpose of this study is to investigate the publication conditions of stem cell therapy for meniscal regeneration and to visualize the research trends and frontiers. Methods: Relevant publications relevant to stem cells for meniscal regeneration was retrieved SCI-Expanded of the Web of Science database from 2012 to 2022. Research trends in the field were analysed and visualized by CiteSpace and VOSviewer. Results: A total of 354 publications were collected and analysed. The United States contributed the largest number of publications (118, 34.104%). Tokyo Medical Dental University has contributed the largest number of publications (34) among all full-time institutions. Stem cell research therapy has published the largest number of researches on stem cells for meniscal regeneration (17). SEKIYA. I contributed the majority of publications in this field (31), while Horie, M was the most frequently cited authors (166). #1 tissue engineering, #2 articular cartilage, #3 anterior cruciate ligament, #4 regenerative medicine, #5 scaffold are the chief keywords. This indicates that the current research hotspot has been transformed from basic surgical research to tissue engineering. Conclusion: Stem cell therapy is a promising therapeutic method for meniscus regeneration. This is the first visualized and bibliometric study to thoroughly construct the development trends and knowledge structure in the research field of stem cell therapy for meniscal regeneration in the past 10 years. The results thoroughly summarize and visualize the research frontiers, which will shed light on the research direction of stem cell therapy for meniscal regeneration.

Tissue-specific regulatory mechanism of LncRNAs and methylation in sheep adipose and muscle induced by Allium mongolicum Regel extracts.

Allium mongolicum Regel (A. mongolicum) is a perennial and xerophytic Liliaceous allium plant in high altitude desert steppe and desert areas. Feeding A. mongolicum greatly reduced unpleasant mutton flavor and improves meat quality of sheep. We analyzed epigenetic regulatory mechanisms of water extracts of A. mongolicum (WEA) on sheep muscle and adipose using RNA-Seq and whole-genome Bisulfite sequencing. Feeding WEA reduced differentially expressed genes and long non-coding RNAs (lncRNAs) between two tissues but increased differentially methylation regions (DMRs). LncRNA and DMR targets were both involved in ATP binding, ubiquitin, protein kinase binding, regulation of cell proliferation, and related signaling pathways, but not unsaturated fatty acids metabolism. Besides, tissue specific targets were involved in distinct functional annotations, e.g., Golgi membrane and endoplasmic reticulum for muscle lncRNA, oxidative phosphorylation metabolism for adipose lncRNA, dsRNA binding for muscle DMRs. Epigenetic regulatory networks were also discovered to discovered essential co-regulated modules, e.g., co-regulated insulin secretion module (PDPK1, ATP1A2, CACNA1S and CAMK2D) in adipose. The results indicated that WEA induced distinct epigenetic regulation on muscle and adipose to diminish transcriptome differences between tissues, which highlights biological functions of A. mongolicum, tissue similarity and specificity, as well as regulatory mechanism of mutton odor.

Molecular and immunological characterizations of interleukin-11 in large yellow croaker (Larimichthys crocea).

Interleukin (IL)-11 is a multifunctional cytokine that exerts a series of important immunomodulatory effects and exists in many tissues and cells. A 1106-bp nucleotide sequence representing the complete cDNA of IL-11 was obtained from large yellow croaker (Larimichthys crocea), containing an open reading frame (ORF) of 603 bp encoding for 200 amino acids (aa). The predicted LcIL-11 protein included a 12aa signal peptide and a conserved IL-11 domain. The polypeptide sequence identities between LcIL-11 and its counterparts in mammals and other fish are from 84% to 92% with known fish IL-11a and 22%-27% with fish IL-11b. LcIL-11 mRNA existed in most tissues with the most predominant expression in the gill. After immune challenge, the expression levels of LcIL-11 were induced largely in vivo and in vitro, with the peak-value of 32 times as much as the control in the liver at 24 h after Vibrio parahaemolyticus injection (p < 0.05) and the greatest value of 13.9 times as much as the control in LCK cells at 12 h after poly I:C stimulation (p < 0.05). Furthermore, the overexpression vector pcDNA3.1-LcIL-11 was constructed and transfected to LCK cells. Our results showed that the transcriptional expression levels of tumor necrosis factor (TNF)-α and myxovirus resistant protein (Mx) significantly up-regulated in LCK cells after LcIL-11 overexpression (p < 0.05). However, no significant changes of IL-1ß, janus kinase (JAK)2 and signal transducers and activators of transcription (STAT)5 was detected. Our finding indicated that LcIL-11 might enhance TNF-α and antiviral protein Mx expression in large yellow croaker.

Identification and expression profiles of ERK2 and ERK5 in large yellow croaker (Larimichthys crocea) after temperature stress and immune challenge.

Fish is highly affected by many environmental stresses such as temperature and invasive infection. The extracellular signal-regulated kinase (ERK) pathway, part of the mitogen-activated protein kinase (MAPK) family, is found to act as crucial mediators for cell differentiation, proliferation and cell response to various stresses. In the present study, ERK2 (LcERK2) and ERK5 (LcERK2) were cloned and characterized from large yellow croaker, Larimichthys crocea. The full length cDNA sequence of LcERK2 was of 1910 bp, including an ORF of 1110bp encoding a polypeptide of 369 amino acids. The full length cDNA sequence of LcERK5 was of 3720bp, including an ORF of 3375bp encoding a polypeptide of 1124 amino acids. Multiple alignments showed that both LcERK2 and LcERK5 contained highly conserved TEY motif and S_TKc domain in MAPK family and the unique catalytic and active structures of ERK2 and ERK5. Subcellular localization revealed that both LcERK2 and LcERK5 expressed in the cytoplasm and cell nucleus. The expression of LcERK2 and LcERK5 were detected in most tissues of large yellow croaker, with the most predominant expression of LcERK2 in brain and LcERK5 in gill, and the weakest expression of LcERK2 in liver and LcERK5 in intestine, respectively. The expression levels of LcERK2 and LcERK5 after temperature stress and poly I:C and flagellin challenge were investigated in LCK (L. crocea kidney) cells. After temperature stress, significant down-regulations of LcERK2 transcripts were detected after 10 °C stress (p < 0.05) whereas LcERK2 transcripts increased significantly after 35 °C stress (p < 0.05). However, significant down-regulations of LcERK5 expression were detected at most time points after both cold and heat stress (p < 0.05). However, significant up-regulations of LcERK2 and LcERK5 transcripts were found after immune challenge (p < 0.05). Our results showed that LcERK2 transcripts enhanced after heat stress and both LcERK2 and LcERK5 transcripts could be induced by immune challenge. These findings indicated that LcERK2 might be more important in fish response to high temperature stress and both LcERK2 and LcERK5 might play an important role in fish immune response.

Characterization and expression analysis of Toll-like receptor 2 gene in large yellow croaker, Larimichthys crocea.

Toll-like receptor 2 (TLR2) plays an important role in innate immune responses. Here we describe the isolation and characterization of the full-length cDNA sequence of toll-like receptor 2 in large yellow croaker Larimichthys crocea (LcTLR2). The LcTLR2 cDNA contains a 5'-terminal untranslated region (5'-UTR) of 135 bp, an open reading frame (ORF) of 2478 bp encoding a polypeptide of 825 amino acid residues and a 3'-UTR of 50 bp. Subcellular localization analysis suggested that the LcTLR2-pEGFP was mainly expressed in cytoplasm. Quantitative real-time reverse transcription PCR (qRT-PCR) analysis revealed a broad expression of LcTLR2 in most examined tissues, with the most predominant expression in blood, followed by spleen, and the weakest expression in stomach. The expression levels of LcTLR2 after injection with Vibrio parahaemolyticus, Lipopolysaccharides (LPS) and poly inosinic:cytidylic (polyI:C) were investigated in spleen, head-kidney and liver. Our results showed that LcTLR2 transcripts increased significantly after all the three immune challenges (p < 0.05). However, compared with polyI:C and LPS, higher expression levels of LcTLR2 were induced in all examined tissues after V. parahaemolyticus stimulation. In addition, the expression levels of LcTLR2 after flagellin, polyI:C, peptidoglycan (PGN) and LPS challenge in LCK were investigated, our findings showed that high LcTLR2 transcripts were induced after flagellin and PGN stimulation, suggesting that LcTLR2 might play a vital role in fish defense against bacterial infection. Furthermore, compared with LPS, flagellin and peptidoglycan might play an important role in LcTLR2 induction in large yellow croaker.

Cloning and expression analysis of interferon regulatory factor (IRF) 3 and 7 in large yellow croaker, Larimichthys crocea.

The interferon regulatory factor (IRF)3 and IRF7 are considered to play essential roles in innate immune system's antiviral responses. In this report, the full-length cDNA and genomic structure and immune response characterizations of IRF3 and IRF7 were investigated in large yellow croaker, Larimichthys crocea. The full-length cDNA of L. crocea (Lc)IRF3 was of 2204 bp, including a 5'-terminal untranslated region (UTR) of 41 bp, a 3'-terminal UTR of 774 bp and an open reading frame (ORF) of 1389 bp encoding a polypeptide of 462 amino acids residues. The full-length cDNA of LcIRF7 was of 1979 bp, including a 5'-terminal UTR of 47 bp, a 3'-terminal UTR of 636 bp and an ORF of 1296 bp encoding a polypeptide of 431 amino acids. The putative amino acid sequence of both LcIRF3 and LcIRF7 contained a typical IRF domain at the N-terminal and an IRF3 domain at the C-terminal. Furthermore, we obtained 4517 nucleotides (nt) LcIRF3 genome sequence based on the full-length cDNA, which contained 11 exons and 10 introns. The full-length genome sequence of LcIRF7 was of 3991 nucleotides, including 9 exons and 8 introns. Quantitative real-time reverse transcription PCR analysis revealed a broad expression of LcIRF3 and LcIRF7 with the most predominant expression of LcIRF3 and LcIRF7 in the liver and in the gill, respectively. The expression levels of LcIRF3 and LcIRF7 after challenged with LPS, poly I:C and Vibrio parahaemolyticus were tested in blood, spleen and liver. The results showed that the highest relative expression of LcIRF3 was in the liver at 24 h after poly I:C injection with 90 times greater than that of the non-injection group (p < 0.05). Moreover, LcIRF3 transcription increased significantly at most time point in blood and spleen tissue after poly I:C stimulation compared with that of the control group. After LPS injection, the peak value of LcIRF7 was in the liver with 207 times (at 3 h) as much as that in the control group (p < 0.05). In addition, LcIRF7 expression was significantly induced by poly I:C injection in spleen. Both LcIRF3 and LcIRF7 transcripts did not show significant change after V. parahaemolyticus stimulation. These results indicated that IRF3 and IRF7 might play an important role in large yellow croaker's defense against viral and bacterial infection.